Download Characterization of novel canine bocaviruses and their association

Journal of General Virology (2012), 93, 341–346
Short
Communication
DOI 10.1099/vir.0.036624-0
Characterization of novel canine bocaviruses and
their association with respiratory disease
Amit Kapoor,1 Natasha Mehta,1 Edward J. Dubovi,2 Peter Simmonds,3
Lakshmanan Govindasamy,4 Jan L. Medina,1 Craig Street,1 Shelly Shields5
and W. Ian Lipkin1
Correspondence
1
Amit Kapoor
2
Center for Infection and Immunity, Columbia University, New York 10032, USA
College of Veterinary Medicine at Cornell, Ithaca, NY 14853, USA
[email protected]
3
Centre for Immunology, Infection and Evolution, University of Edinburgh, Edinburgh EH9 3JT, UK
4
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610,
USA
5
Pfizer Veterinary Medicine Research and Development, New York 10017, USA
Received 21 July 2011
Accepted 21 October 2011
We report the first identification, genetic characterization and disease association studies of
several novel species of canine bocaviruses (CBoV). Evolutionary analysis confirmed that CBoV
are genetically distinct from the only other known canine bocavirus, minute virus of canines, with
which they share less than 63, 62 and 64 % protein identity in NS, NP and VP genes, respectively.
Comparative genetic analysis of 37 VP gene variants found in diseased and healthy animals
showed that these novel viruses are genetically highly diverse and are common in canine
respiratory infections that have remained undetected until now. Interestingly, we observed that a
CBoV genotype with a unique deletion in the VP2 gene was significantly more prevalent in animals
with respiratory diseases compared with healthy animals.
Parvoviruses, which frequently infect animals through the
faecal–oral route, are small, non-enveloped icosahedral
viruses with linear ssDNA genomes (Fauquet et al., 2004).
They are members of the family Parvoviridae, which
comprises two subfamilies, Densovirinae and Parvovirinae,
members of which infect non-vertebrate and vertebrate
hosts, respectively (Brown, 2010; Fauquet et al., 2004). The
International Committee on Taxonomy of Viruses (ICTV)
has further classified the subfamily Parvovirinae into six
genera: Dependovirus, Bocavirus, Erythrovirus, Parvovirus,
Amdovirus and Partetravirus. Bocaviruses are unique
among parvoviruses as they contain a third ORF between
the non-structural- and structural-coding regions (Kapoor
et al., 2010b; Manteufel & Truyen, 2008; Qiu et al.,
2007). The genus Bocavirus currently includes the bovine
parvoviruses (BPV), minute virus of canines (MVC)
(Fauquet et al., 2004), porcine bocaviruses (Cheng et al.,
2010), gorilla bocavirus (GBoV) (Kapoor et al., 2010a) and
four species of human bocaviruses (HBoV 1–4) (Allander
et al., 2005; Arthur et al., 2009; Chieochansin et al., 2007;
Kapoor et al., 2009, 2010b). Bocaviruses commonly infect
the respiratory and gastrointestinal tract of young animals
The GenBank/EMBL/DDBJ accession numbers for the sequences
reported in this paper are JN648103 and JN648104–JN648139.
A supplementary table is available with the online version of this paper.
036624 G 2012 SGM
and humans, and except for BPV, the pathological
manifestations of these infections remain largely unknown
(Don et al., 2011; Kapoor et al., 2011; Manteufel & Truyen,
2008; Martin et al., 2009, 2010).
First discovered in 1967 in faeces of healthy dogs, MVC is
the only known bocavirus that infects dogs. It can cause
abortions in bitches and severe respiratory infections in
newborn puppies, but infections are mostly subclinical in
adult animals (Carmichael et al., 1991). MVC replicates to
high titres in Walter Reed/3873D (WRD) canine cells,
making it a useful model system to dissect the replication
kinetics of genetically similar, but uncultivable, HBoV (Sun
et al., 2009). During a metagenomic study conducted to
better characterize the respiratory viral flora of domestic
animals, we observed several sequences with distant
amino acid sequence similarity to animal parvoviruses in
respiratory samples from diseased dogs. Extension of these
novel sequences using a primer walking approach revealed
the presence of a novel bocavirus tentatively named canine
bocavirus (CBoV). Thereafter, a consensus PCR assay using
primers targeting conserved structural protein motifs was
used to determine the prevalence and genetic diversity of
CBoV variants in a cohort of respiratory samples obtained
from diseased and healthy dogs (Supplementary Table S1,
available in JGV Online). Briefly, extracted nucleic acids
from each sample were used for two rounds of nested PCR
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A. Kapoor and others
(first round: forward-CBoV-QFX1-f1, 59-CARTGGTAYGCTCCMATYTTTAA-39 and reverse-CBoV-QFX1-r1, 59-TGGCTCCCGTCACAAAAKATRTG-39; and second round:
forward-CBoV-QFX1-f2, 59-TGGTAYGCTCCMATYTTTAAYGG-39, reverse-CBoV-QFX1-r2 59-GCTCCCGTCACAAAAKATRTGAAC-39). The amplification products (~400
nt long) representing the partial CBoV VP1 gene were
sequenced for confirmation and to determine viral genetic
diversity. Of the 158 animals tested, 36 (23 %) were infected
with CBoV variants (Supplementary Table S1).
The nearly complete genome of CBoV variant con-161 is
5413 nt (GenBank accession no. JN648103) and bears a
high degree of similarity to other known bocaviruses predicted to contain non-coding terminal sequences flanking
the three large ORFs (Fig. 1). ORF1 encodes a 648 aa nonstructural (NS) protein. ORF2 encodes 712 aa overlapping
the VP1/VP2 capsid ORFs. ORF3 encodes a 195 aa NP1
protein. The non-coding region on left-hand side (LHS)
terminus, located at the 59 end of positive-sense ssDNA
genomes or at the amino terminus of NS protein, is 306 nt.
Its secondary structure folds into an imperfect palindrome
and contains a rabbit-ear structure similar to MVC and
BPV (Fig. 1). The right-hand side (RHS) non-coding
region, found at the 39 end of positive-sense ssDNA
genomes, is not identical to the LHS terminus and forms
an imperfect palindrome. The LHS terminus showed
highest sequence similarity to the MVC terminus and also
contained a NS-binding site (Sun et al., 2009). While the
MVC and BPV NS protein-coding regions encode a single
long NS protein, recent studies have shown that the
homologous region of all four HBoV species encodes two
NS proteins of variable length (Chen et al., 2010; Kapoor
et al., 2010a). Remarkably, although CBoV is genetically
similar to HBoV in NS gene splicing, it is most similar to
-3´
MVC. The CBoV NS-coding region encodes a shorter NS
protein, as well as conserved RNA splicing signals essential
to generate a longer NS protein (Fig. 1). Like other
bioinformatics analyses and predictions, these observations require experimental validation in subsequent
studies.
To determine CBoV’s appropriate phylogenetic classification and genetic relatedness to other known parvovirus
species, at least one representative virus, as well as the
reference genome from each human and animal bocavirus
species and their translated protein sequences, was used for
generating sequence alignments. The most appropriate
protein or nucleotide substitution model was determined
using MEGA, and the method with lowest scores was used to
calculate pair-wise distances and to construct phylogenetic
trees (Fig. 2). All three CBoV proteins (NS, NP and VP)
were genetically most related to corresponding MVC proteins; however, there was more genetic diversity/variability
between CBoV and MCV proteins than among different
HBoV species or different species of genus Parvovirus (Fig.
2). The ICTV criteria for species classification within the
genus Bocavirus specify that members of each species are
probably antigenically distinct and that natural infection is
confined to a single host species. Species are defined as
having ,95 % homologous NS gene DNA sequences (http://
www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/). While the antigenic properties of CBoV were not studied here, we found
.35 % genetic divergence in the NS protein compared with
other known bocaviruses, suggesting that CBoV and its
variants represent one or more novel species within the genus
Bocavirus.
Parvoviral capsid proteins contain determinants of immunogenicity and host cell tropism. Minor genetic changes in
-5´
CBoV
BPV
MVC
-3´
-5´
MVC
CBoV
HBoV
GBoV
-3´
-5´
BPV
Fig. 1. Comparative genomic organization and LHS inverted terminal repeat structure of different bocavirus species. All
genomes were aligned starting from first N-terminal amino acid codon of the NS gene (nucleotide positions) to comparatively
show the location of the putative RNA splicing region in the NS exon (shown as filled triangle).
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Novel canine bocaviruses
Fig. 2. Phylogenetic analyses of inferred amino acid sequences of the three principal ORFs (NS, NP and VP proteins) of human
and animal bocaviruses; bootstrap values of .70 % are shown. Bars, represent 0.1 substitutions per amino acid site.
these proteins are known to alter the host range and
pathogenic potential of parvoviruses (Hoelzer et al., 2008a,
b; Parrish & Kawaoka, 2005). Moreover, evolutionary
studies confirmed that, unlike other DNA viruses, parvoviruses can evolve rapidly displaying frequent recombination and mutation rates that approach the high
mutation rates observed in RNA viruses (Shackelton &
Holmes, 2006; Shackelton et al., 2005, 2007). Expecting
that genetic diversity in CBoV capsid proteins should
influence their pathogenic potential, we classified CBoV
variants according to the genetic relatedness in their capsid
protein sequences (Fig. 3a). Calculation of pair-wise
distances using the 261 nt long VP1/2 region of 35 CBoV
variants resulted in up to 37 % (mean diversity of 15 %)
nucleotide and 17 % (mean diversity of 7.3 %) protein
sequence divergence (GenBank accession nos JN648104–
JN648139). Phylogenetic analysis suggested that most
CBoV variants can be divided into three major genetic
groups, provisionally named CBoV-A to -C, while some
were outliers (Fig. 3a). Our results imply that CBoV represents a highly diverse group of novel canine bocaviruses.
The extent of genetic diversity observed among CBoV
variants characterized in this single study exceeds the
maximum genetic diversity known to exist among all MVC
variants reported worldwide to date (Fig. 3a). Combined
analyses of genetic diversity and PCR prevalence data
suggest that CBoV variants of group A were significantly
http://vir.sgmjournals.org
more prevalent in healthy dogs raised in controlled
environments than in animals from other groups; and
CBoV variants of group C were substantially more
prevalent in dogs with respiratory diseases than in healthy
animals (Supplementary Table S1).
To further investigate genetic diversity between CBoV-A
and -C viruses, we acquired the complete capsid gene
sequence of its representative variants. The CBoV-A
capsid protein showed 89 % protein identity to CBoV-C
(GenBank accession no. JN648135) over its entire length
(714 aa, data not shown). To determine the location of
amino acid position changes on viral capsid structure, we
modelled the secondary structure of CBoV-A and -C for
comparison. A homology model was made by giving a
human B19 crystal structure coordinates (PDB ID:1S58) as
a template model with knowledge based protein modelling
program, SWISS-MODEL (Arnold et al., 2006). The final
model was structurally and geometrically consistent and
did not reveal either structure or sequence discrepancies.
The model geometries were cross-validated with the
PROCHECK program (Laskowski et al., 1996) to check the
accuracy of the CBoV homology model. Both B19 and
CBoV structures were superimposed and resulted in a
good r.m.s.d value of 0.7 Å using the COOT program
(Emsley & Cowtan, 2004). The graphical images were
generated with the PYMOL program (www.pymol.org). The
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A. Kapoor and others
Fig. 3. (a) Genetic diversity of structural proteins among CBoV variants found in diseased (Dis-disease) and healthy (Concontrol) dogs. For comparison, genetic diversity among MVC variants was included in the analysis (lower branch of the tree);
and (b) comparative secondary structure of the capsid protein of CBoV variants was used to decipher the structural changes
caused by insertion of six amino acid residues. In the ribbon diagram of CBoV1, the secondary structure elements (b-strand in
yellow, helix in red and loop in green) are coloured differently. The icosahedral symmetry axes are represented as oval, triangle
and pentagon. In the coil representation of CBoV capsid structure the CBoV-A and -C are shown as red and green,
respectively. Bars, represent 0.05 substitutions per amino acid site.
CBoV model contains a high structurally conserved bbarrel motif, a single a-helix and several stretched loops
adopt a different conformational position (Fig. 3b). The
high structurally similar b-barrel motif region has been
implicated for genome packaging and protecting the virus
capsid from the environmental damage. The fivefold pore
region of the capsid is critical for VP1 externalization,
packing of genome and capsid assembly functions (Gurda
et al., 2010). The intertwining or flexible loops in the CBoV
capsid decorate in the exterior surface region of the capsid
and are highly distinguished from all other parvoviruses.
The variable loops are important to control various
biological properties of the capsid and that includes tissue
tropism, transduction and receptor binding (Gurda et al.,
2010). We noticed that the six amino acid insertion unique
to CBoV-A was located in the variable exposed loop (Fig.
3b). Moreover the structural folding of all four outer
surface exposed loops was very different between CBoV-A
and -C variants possibly reflecting difference in their
biological properties.
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To conclude, we report several previously uncharacterized
species of canine bocaviruses, their sequences, genomic
characteristics and genetic diversity. We also compared the
genetic diversity and the differences in prevalence of these
novel viruses in sick and healthy animals. Animals infected
with CBoV-B1 variants and suffering from respiratory
infections were housed in a shelter facility. Animal shelters
often house animals likely to have a wide variety of
infections (Steneroden et al., 2011). These crowded conditions probably facilitate a higher prevalence of viruses in
shelter animals compared with the healthy pet population
(Helps et al., 2005). Moreover, we could not rule out the
possibility that respiratory diseases in these animals were
caused by other pathogenic respiratory viruses, but even
then the higher prevalence of CBoV-B1 variants in this
population alone suggests that these viruses are more likely
to infect diseased animals (opportunistic infections) or that
they can cause or enhance the pathology of other infections
(co-infections). We note that the HBoV species include
several highly divergent viruses (Kapoor et al., 2010b) and
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Journal of General Virology 93
Novel canine bocaviruses
therefore more comprehensive disease-association studies
should consider the genotype-specific prevalence pattern of
these viruses. Comparable and high genetic diversity among
CBoV variants makes it a more appropriate model to study
HBoV disease-association as well as the evolution and
pathogenicity of parvoviruses. Despite distant phylogenetic
relatedness, we noticed many similarities between CBoV and
HBoV species. Both groups of recently identified viruses are
genetically diverse and contain many species and genotypes
whose pathogenic potential remains unknown (Kapoor
et al., 2010b). Unlike other animal bocaviruses, CBoV and
HBoV have splicing signals in the NS gene and are likely to
encode more than one NS protein (Kapoor et al., 2010a).
Unfortunately, the studies for complete biological characterization of HBoV pathogenesis are hampered by the lack
of a successful cell culture or animal model (Kapoor et al.,
2011). Elucidation of the nearly complete CBoV genome, its
genetic diversity and prevalence will help to establish a
successful cell culture system for these viruses.
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Acknowledgements
We thank Natasha Qaisar for excellent technical assistance. We are
grateful for high throughput sequencing support provided by Roche
454 Life Sciences. This work was supported by awards from the
National Institutes of Health (AI090196, AI081132, AI079231,
AI57158, AI070411, AI090055, AI072613 and EY017404), the
Defense Threat Reduction Agency and USDA 58-1275-7-370.
Kapoor, A., Slikas, E., Simmonds, P., Chieochansin, T., Naeem, A.,
Shaukat, S., Alam, M. M., Sharif, S., Angez, M. & other authors
(2009). A newly identified bocavirus species in human stool. J Infect
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Qaisar, N., Delwart, E. & Lipkin, W. I. (2010a). Identification and
characterization of a new bocavirus species in gorillas. PLoS ONE 5,
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Kapoor, A., Simmonds, P., Slikas, E., Li, L., Bodhidatta, L., Sethabutr, O.,
Triki, H., Bahri, O., Oderinde, B. S. & other authors (2010b). Human
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